Cessation of milk removal leads to changes in the mammary secretion and to initiation of the process of active mammary involution. This process comprises an extensive and highly ordered sequence of changes in tissue and milk composition, which occur during the transition between the lactating and the non-lactating states. The initial stage of mammary involution is triggered by local stimuli that initiate apoptosis, but involution can be reversed by reinitiating milk removal (Capuco and Akers, 1999. J. Mammary Gland Biol. Neoplasia 4:137-144; Wilde et al., 1999. J. Mammary Gland Biol. Neoplasia 4:129-136). This local control can cause involution when milk stasis is induced in individual glands, as was observed in lactating goats following unilateral cessation of milking (Quarrie et al., 1994. Biochem. Soc. Trans. 22:178 S), or in lactating mice following teat sealing (Li et al., 1997. Proc. Natl. Acad. Sci. U.S.A. 94: 3425-3430; Marti et al., 1997. Eur. J. Cell. Biol. 73:158-165).
The further stage of involution is persistent, and milk removal cannot cause resumption of milk secretion (Capuco and Akers, 1999. ibid; Wilde et al., 1999. ibid). Reversal of the persistent state of involution can occur only in a subsequent lactating period after giving birth. This after-parturition stage is characterized by activation of proteases that destroy the lobular-alveolar structure of the gland by degrading the extracellular matrix and basement membrane, as well as by massive loss of alveolar cells.
In the modern dairy industry, lactating animals in herds go through controlled cycles of milking and pregnancy, as such regimes contribute to a significant increase in milk production. In current management of dairy herds, for example cows and goats, there is a significant overlap between lactation and pregnancy, wherein a “dry period” is imposed between 35 to 75 days prior to parturition by cessation of milking. This regime is set to compromise between the need to induce involution, a necessary process for subsequent healthy lactating period, and the requirement for high milk production all year long.
Breastfeeding, or nursing, has been identified as the ideal method of feeding and nurturing infants and is considered a primary factor in achieving optimal infant and child health, growth, and development. Epidemiologic research shows that human milk and breastfeeding of infants significantly decreases the risk for or severity of a large number of acute and chronic diseases, and there are also a number of studies that indicate possible health benefits for the nursing mothers.
With these health benefits, as well as social and economic advantages, the American Academy of Pediatrics and the World Health Organization (World Health Organization. (2003). Global strategy for infant and young child feeding. Geneva, Switzerland: World Health Organization and UNICEF. ISBNO. 9241562218) has issued a policy statement that recommends breastfeeding exclusively for the first six months, then gradually introducing solid foods and continuing to breastfeed for at least six more months and after that for as long as mutually desired. In normal breastfeeding practices, weaning an infant from breastfeeding to another food source such as solid food, formula or fruit juices is a gradual process. As the infant's diet incorporates other food sources, the mother continues to nurse but either decreases the amount of time at each feeding, decreases the number of breast feedings each day and/or slowly increases the time between breast feedings. Under these conditions, the mother's milk naturally diminishes at a slow rate as the infant's demand lessens.
There are circumstances, however, which either prevent a mother from breastfeeding or require that breastfeeding be discontinued abruptly. Some mothers prefer to bottle-feed rather than breastfeed. The death of an infant results in a mother's sudden halt to breastfeeding. Breastfeeding mothers who become pregnant may be advised to discontinue nursing, especially if the risk for miscarriage is high. Abrupt weaning is recommended for the infant with galactosemia; the infant whose mother uses illegal drugs (American Academy of Pediatrics, Committee on Drugs, 1994); the infant whose mother has untreated active tuberculosis, and the infant whose mother has been infected with the human immunodeficiency virus (American Academy of Pediatrics, Committee on Pediatric Aids, 1995). Whether a mother chooses not to breastfeed at all or to discontinue breastfeeding, lactation will continue for a time. For women who are producing breast milk but not nursing, the milk stasis is associated with swelling of the breast to an extent that may cause conspicuous agony, both physically and psychology.
The cessation of milking or nursing is also associated with increased risk of developing mastitis, a disease caused by intramammary infection (IMI) with pathogens, mostly bacteria, but also yeast, fungi, or even algae. Mastitis can be clinical, with local (and in some cases general) clinical signs and milk abnormalities, or subclinical with production losses and lowered milk quality.
It has been shown that injection of crude preparation of casein hydrolyzate (CNH) comprising proteose-peptones (PPs, also known as casein phosphopeptides, CPP) into the udder of a goat or a cow mimics the natural phenomenon of involution, inducing a local inflammatory response and loss of tight junction (TJ) integrity, followed by rapid drying-off of mammary secretion (U.S. Pat. No. 6,391,849; Shamay et al., 2002 Life Sci. 70:2707-2719; Shamay et al., 2003. J. Dairy Sci. 86:1250-1258). The process induced by CNH was more rapid than that induced at natural drying-off. It was further shown that a pure β-casein (β-CN) fraction 1-28 down-regulates milk secretion in cows and goats. The activity of this peptide was correlated with its ability to block potassium channels in the apical membranes of mammary epithelia (Silanikove et al., 2000. Life Sci. 67:2201-2212).
European Patent Application No. EP1375513 discloses that among the peptides derived from casein, peptides having amino acid sequences comprising plural phosphoserine residues show a strong immuno-enhancing activity. Specifically, the invention relates to an immuno-enhancing agent comprising a peptide consisting of the amino acid sequence Q1-SerP-X-SerP-Q2, wherein, SerP represents the phosphoserine residue, X represents one to three of any amino acid residues, and Q1 and Q2 are independently absent or represents at least one of any amino acid residue.
The inventors of the present invention and co-workers have previously disclosed pharmaceutical compositions comprising casein-derived peptides in the form of a ready to use, sterile, clear solution, substantially devoid of micelles and having a pH above 6.0 (International Patent Application Publication No. WO2006/117784). It was suggested that the minimal active peptide comprises the sequence Ser(p)-Ser(p)-Ser(p)-Glu-Glu. However, these pharmaceutical compositions comprise a plurality of peptides obtained by hydrolysis of casein.
Thus, there is a recognized need for, and would be highly advantageous to have pharmaceutical composition comprising peptides which are defined by their amino acid sequence and concentration. Such composition can be produced on a commercial scale in a reproducible manner.